We propose a Resource for Native Mass Spectrometry Guided Structural Biology that will be led by a team of scientists including experts in MS instrumentation (Wysocki, OSU; Russell, Texas A&M), separation science coupled to ionization (Olesik, OSU; Badu, OSU; Holland; WVU), and computational chemistry (Lindert, OSU). The goal of this Resource is to build and validate an integrated workflow for structural characterization of protein:protein, membrane protein:lipid, and protein:RNA complexes that are critical for an array of cellular and organismal processes. There is a growing appreciation of the pivotal role and utility of MS-based approaches for structural characterization of biomacromolecules, filling critical gaps and complementing other structural biology tools. Thus, our workplan leverages innovative MS methods to determine: (i) m/z of all binding partners and the intact complex, (ii) component stoichiometry, (iii) heterogeneity, if present, (iv) the relative topology/architecture/ conformational diversity of components in the complex, and (v) the hierarchy of assembly, including binding affinities of individual subunits. Investigators from across the United States (in WA, OR, UT, CA, AZ, TX, TN, OH, MD, MA) and international sites will contribute challenging biomedical projects on topics including viral hemorrhagic fevers, HIV, cataract formation, and neurological disorders. Our workflow is designed to advance understanding of: (i) how formation of and dynamic changes in wideranging macro-molecular assemblies determine their biological roles, (ii) how alterations in assembly/architecture of these complexes lead to disease, and (iii) foundational principles for building synthetic mimics needed for biomedical applications. In this Resource, ten Driving Biomedical Projects (DBPs) provide biomedical structural characterization challenges and serve as drivers and test beds for five Technology Research and Development (TR&D) projects. The TR&Ds provide: (i) effective separation methods to purify and deliver native macromolecular complexes well suited for MS, (ii) effective surface-induced dissociation and UV-photodissociation technologies, (iii) measurement of the intact complexes and their non-covalent (sub-complex) and covalent dissociation products with high resolution ion mobility (IM) and/or MS, and (iv) computational tools for structure prediction. Computational tools will use restraints from surface-induced dissociation patterns and collision cross sections from IM-MS experiments, and from MS-based solution measurements (H/D exchange and covalent labeling). Existing ties between the TR&Ds and instrument companies (Waters, Thermo, Bruker, Sciex, Phenomenex) as well as a national laboratory (PNNL) will aid technology development and expedite technology dissemination. Our training and dissemination activities (e.g., workshops, beta device installations) are designed to increase outreach and maximize Resource payoffs.